The generation of complete or nearly complete eukaryotic genome sequences has produced an explosion of insights into the coding potential and evolution of genomes. However, we have less information about how key processes involving DMA are regulated, including transcription, replication, repair, and chromosome segregation. DMA sequence alone cannot be expected to reveal the mechanisms involved in genome regulation and inheritance, because these processes do not act on 'naked'DMA. In the context of the cell, DMA is packaged as chromatin, whose composition and organization regulate the accessibility and function of DMA sequences. Chromatin displays increasingly complex levels of organization and composition, starting with the basic nucleosome unit and progressing through higher order structures. Chromatin organization is critical for utilizing information stored in the genome;failure to accurately target or maintain chromosomal proteins and chromatin components results in aberrant patterns of gene expression and chromosome behavior, and is associated with many human diseases, most notably cancer. We propose to participate in the MODENCODE project by determining the locations of 125 chromosomal proteins and histone modifications across the Drosophila melanogaster genome. The proteins and modifications under study are involved in basic chromosomal functions such as DNA replication, gene expression, gene silencing, and inheritance. We will perform Chromatin ImmunoPrecipitation (ChIP) with antibodies obtained commercially and generated and validated by this project, isolate and label the precipitated DNA, and apply the probes to genomic tiling arrays. Data generated by scanning the hybridized arrays will be analyzed by statistical methods, and the array data will be validated by independent analyses in cells and animals. We will initially assay localizations using chromatin from three cell lines and two embryonic stages, and will then extend the analysis of a subset of proteins to four additional animal tissues/stages. We will then perform a variety of comparisons between protein 'landscape'data sets, including analyses of combinatorial patterns of modifications and chromosomal proteins, tissue-specific differences, and interactions among proteins involved in the same epigenetic pathways. Finally, all validated data and analyses will be made available to members of the ENCODE project and scientific community. Successful completion of this project will provide basic information about the distributions of chromatin components across the Drosophila genome sequence, which will serve as a foundation for future functional studies. In addition, the data and analysis are highly likely to provide information critical to understanding the roles of chromatin in human cells and diseases.